THE ECOLOGY OF WS COOPERATION: AN EMERGENT GROUP-LEVEL PHENOTYPE

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A TEST OF WS COOPERATION USING THE HAYSTACK MO

We are beginning to see that the awesome wonder of the evolution from amoeba to man – for it is without a doubt an awesome wonder – was not the result of a mighty word from a creator, but of a combination of small, apparently insignificant processes. The structural change occurring in a molecule within a chromosome, the result of a struggle over food between two animals, the reproduction and feeding of young – such are the simple elements that together, in the course of millions of years, created the great wonder. This is nothing separate from ordinary life. The wonder is in our everyday world, if only we have the ability to see it.

INTRODUCTION

Previous work by Rainey and Rainey (2003) showed that the WS phenotype satisfies the criteria for being considered a cooperative trait, a trait that evolved de novo in the laboratory (Section 1.4.1.1). Firstly, there is a cost to cooperation, because WS types have a relative fitness of 0.8 relative to ancestral SBW25 when incubated statically with abundant resources (Rainey and Rainey, 2003). Secondly, this cost is offset by benefits provided by an emergent group-level phenotype – the mat occupying a novel niche at the air-liquid interface, as evidenced by the increase in frequency of WS types in a structured environment as oxygen becomes limiting. Thirdly, selfish ancestral-like SM types arise in microcosms founded by WS types that are fitter in the presence of WS types than in their absence. Finally, the emergent group-level phenotype (i.e. the mat) is destroyed as the number of defecting SM types increases too high in frequency – a ‘tragedy of the commons’ (Hardin, 1968).
MLS 1 theory predicts that under certain conditions, group structure can favour the evolution and maintenance of cooperative individuals (Wright, 1945; Wilson, D.S., 1975; Damuth and Heisler, 1988; Sober and Wilson, 1998; Okasha, 2006a). The Haystack model (Maynard Smith, 1964), has commonly been used to describe the procedure for experimental studies in an MLS 1 framework. In the Haystack model, it is supposed that a fictitious population of mice live entirely in haystacks. A haystack is always colonised by a single fertilised female and her offspring live in the haystack until the next year (one group-generation¹). At the next year, new haystacks become available, and the mice disperse, mate and compete to colonise the newly available haystacks. Maynard Smith introduced the idea to the literature for the purpose of distinguishing ‘group selection’ (i.e. differential proliferation and extinction of groups) from kin selection to show that ‘group selection’ of this sort was “too artificial to be worth pursuing further” (Maynard Smith, 1964, p.1146). However, his original model in 1964 was a mixture of MLS 1 and MLS 2 concepts, and assumed a worst-case scenario that is biologically restrictive – that cooperative types would always become extinct within mixed groups (haystacks) of cooperators and selfish types before the end of the group-generation. This assumption has been relaxed in future examples of the Haystack model to be more biologically realistic and to fit precisely into an MLS 1 framework, such that cooperators only decrease in proportion within a group during a group-generation (Wilson, 1987). Therefore, the experimental procedure that is described by the Haystack model, in an MLS 1 framework predicts that, while cooperators are less fit within a group, they may increase in frequency within the overall population if groups with more cooperators contribute a disproportionately high number of individuals to the next group-generation (Damuth and Heisler, 1988; Sober and Wilson, 1998; Gould, 2002; Okasha, 2006a). In essence, MLS 1 theory predicts that group structure will increase the spread of a cooperative trait through a population whenever there is a positive relationship between the number of cooperators and the number of individuals within a group.
The standard experimental microcosm can be exploited as an investigative tool to test the Haystack model, because it is a simple and effective way of dividing populations of cooperators and selfish types into groups¹ (the haystacks). To test the Haystack model in an MLS 1 framework, the phenotypic characteristics of individuals must be measured, and the fitness of those individuals must depend on their organisation into groups. Individuals within each group (microcosm) may be either cooperators (WS types) or selfish types (SM types). All groups within a population are founded with ancestral SBW25 (SM type), and therefore all WS types arise de novo. Crucially, stochastic processes guarantee that, by chance, different WS genotypes will arise in each microcosm and, that different microcosms will vary in their proportion of WS types. Thus, group membership will differentially affect the fitness of individuals within different groups. As in the Haystack model, group structure can periodically be removed to allow the mixing of individuals from different groups (microcosms). This affords an opportunity to individuals within more successful groups to contribute disproportionately to the next generation of groups (MLS 1). The proportion of WS individuals in a population with this group structure and dispersal (experimental treatment) can be contrasted with the proportion of WS individuals in a population in which individuals from different groups are never afforded the opportunity to mix (control treatment). This provides us with an experiment to investigate the hypothesis that group-structure enhances cooperation among the WS types of the P. fluorescens system.
This work extends the results of Rainey and Rainey (2003) by studying the dynamics of the P. fluorescens system to achieve three main aims. Firstly, SBW25 and the neutrally marked SBW25-lacZ strain were grown statically for ten days to verify that the diversification of genotypes via adaptive radiation is conserved for the marked strain (Section 3.2.1). The diversification pattern of genotypes was used to determine the appropriate durations for the group-generations of the selection regime (72 h and 24 h) to use in the multi-level selection experiments. Secondly, the WS system was used to test the Haystack model using a 72-h group-generation time, which predicts that the cooperative WS types will be favoured when groups are afforded the opportunity to mix (Section 3.2.2). Thirdly, the group-generation time was shortened to 24 h so that SM types were favoured within each microcosm to test the Haystack model under conditions that were expected to disfavour the evolution of cooperative WS types (Section 3.2.3).

RESULTS

THE DIVERSIFICATION OF NEUTRALLY MARKED SBW25-LACZ IS CONGRUENT WITH WILD-TYPE SBW25

fluorescens SBW25 rapidly diversifies in spatially structured microcosms into a variety of niche-specialist genotypes (Rainey and Travisano, 1998). A marked variant, SBW25-lacZ, had been shown to have no measurable cost associated with the marker on multiple substrates, and in planta (Zhang and Rainey, 2007). In addition, the marked strain SBW25-lacZ was grown under continuous, shaking, log-phase growth conditions for ten days to verify its long-term neutrality with respect to fitness (Appendix 9.1.1). Imperative to long-term experiments in which strains have time to evolve and adapt is that the marked strain shows no deviation in its ecology and although SBW25-lacZ was expected to undergo a similar adaptive radiation in a spatially structured environment, this had never been shown. Therefore, by way of a preliminary experiment, it was crucial to show that the ecology of this adaptive radiation was the same in both SBW25 and SBW25-lacZ. Each strain was inoculated separately into eight replicate lines of static microcosms and left to grow for between one and ten days. At each 24-h time point, the microcosms were sampled destructively by vortexing, diluted and plated on KB plates (or KB+Xgal). The comparison between the adaptive radiations of SBW25 (white colonies) and SBW25-lacZ (blue colonies) is shown in Figure 3-1.

MLS 1 WITH 72-H GROUP-GENERATION TIME

Two populations, each consisting of 12 groups (microcosms), were studied in a long-term experiment totalling 36 days of experimentation (Section 7.2.16). Each group (microcosm) was founded with an SM type (one with SBW25 and the other with SBW25-lacZ) and incubated statically for 72 h. One of the populations was evolved under the experimental treatment (Figure 3-3) and the other population was evolved as a control experiment (Figure 3-4). The difference between the experimental and the control treatments is the opportunity for competition among groups. In the experimental treatment, equal volumes of each the 12 groups were mixed together following the 72-h static incubation. Samples were taken to estimate the proportion of WS types in the mixed population and 12 new groups were founded from the mixed population to begin the next group-generation. The ten-day adaptive radiation experiment showed that 72 h of static incubation in a microcosm is sufficient to favour the evolution of WS (Figure 3-1). Therefore, WS types were expected to evolve within each microcosm within the first group-generation.

1 INTRODUCTION 
1.1 EVOLUTION: THE UNITY OF LIFE
1.2 COOPERATION
1.3 THE POWER OF MICROBIAL MODEL SYSTEMS
1.4 THE P. FLUORESCENS EXPERIMENTAL SYSTEM
1.5 P. FLUORESCENS EVOLUTIONARY GENETICS
1.6 SUMMARY
2 VARIATION AMONG INDEPENDENT WRINKLY SPREADER GENOTYPES 
2.1 INTRODUCTION
2.2 RESULTS
2.3 DISCUSSION
3 A TEST OF WS COOPERATION USING THE HAYSTACK MODEL 91
3.1 INTRODUCTION
3.2 RESULTS
3.3 DISCUSSION
4 THE ECOLOGY OF WS COOPERATION: AN EMERGENT GROUP-LEVEL PHENOTYPE 
4.1 INTRODUCTION
4.2 RESULTS
4.3 DISCUSSION
5 SELECTION FOR GROUP REPRODUCTION VIA A DEVELOPMENT-LIKE PROCESS 
5.1 INTRODUCTION
5.2 RESULTS
5.3 DISCUSSION
6 FINAL DISCUSSION 
6.1 OVERVIEW OF MAIN RESULTS
6.2 FUTURE DIRECTIONS
6.3 FINAL COMMENT
7 MATERIALS AND METHODS 
7.1 MATERIALS
7.2 METHODS
8 REFERENCES
9 APPENDICES 
9.1 APPENDIX ITEMS FROM CHAPTER
9.2 APPENDIX ITEMS FROM CHAPTER 2
9.3 APPENDIX ITEMS FROM CHAPTER 3
9.4 APPENDIX ITEMS FROM CHAPTER 4
9.5 APPENDIX ITEMS FROM CHAPTER 5
9.6 APPENDIX ITEMS FROM CHAPTER 6
GET THE COMPLETE PROJECT
The Evolution of Cooperation: Insights from Experimental Populations of Pseudomonas fluorescens